American Journal of Plant Sciences, 2011, 2, 35-42
doi:10.4236/ajps.2011.21004 Published Online March 2011 (http://www.SciRP.org/journal/ajps)
35
Effects of Sea Salts on Induction of Cell
Proliferation in Liquid Cultures of Mangrove
Plants, Sonneratia caseolaris and S. alba
Raiki Yamamoto1, Yoshifumi Kawana1, Reiko Minagawa2, Hamako Sasamoto2
1Graduate School of Environment and Information Sciences, Yokohama National University, Yokohama, Japan; 2Faculty of Envi-
ronment and Information Sciences, Yokohama National University, Yokohama, Japan.
Received December 1st, 2010; revised December 13th, 2010; accepted December 29th, 2010.
ABSTRACT
The effects of five salt ingredients of sea water, KCl, NaCl, CaCl2, MgCl2 and MgSO4, on induction of cell pro liferation
in Sonneratia caseo laris were investigated. Proliferation was examined in tissue explants derived from such as leaves,
cotyledons, and hypocotyls using a small-scale liquid culture method. Ad dition of 12.5-25 mM of MgCl2 was unique in
stimulating cell proliferation in all tissues of S. caseolaris. Otherwise, differen t effects of salts were ob served among the
three tissues. In hypocotyl culture, 25-50 mM of NaCl and CaCl2 stimulated cell divisions. Tolerance to 100 mM of
MgSO4 was observed in leaves. Three osmotica lly active compounds commonly used in tissue cultu re, sorbitol, manni-
tol and glycinebetain e, were also tested to assess the importance of osmotic effects on cell proliferatio n. No significant
stimulation by these was observed over a wide range of concentrations. Data were compared with those of cotyledon
cultures of another mangrove, S. alba, which exhibits no stimulation by MgCl2, stimulation by KCl and tolerance to
NaCl. Mechanisms for adaptation of mangrove plants to various and high salts were discussed by comparing the dif-
ferences in reaction to salts in cultures of two Sonneratia mangrove species of the same genera growing different salt
environment.
Keywords: Halophilic, Ions, Salt Tolerant, Sonneratiaceae, S. caseolaris
1. Introduction
Mangrove plants are mainly tree species that grow in
tropical and subtropical brackish coastal regions. This
group includes more than 100 species from different
families [1]. Micropropagation of these species is needed
for conservation of mangrove forests threatened by po-
tential destruction [2]. Biotechnological breeding tech-
niques such as somatic hybridization of trees [3,4], may
be promising tools for a year-round supply to replenish
saplings and protect salty soil environment from deserti-
fication [5].
Studies on salt injury to crops are an increasingly im-
portant topic as soil damage from salt accumulation is
proceeding [6]. Clarifying salt-tolerant mechanisms in
cell and tissue culture systems of mangrove plants, and
introduction of their characteristics through somatic hy-
bridization or genetic transformation into crop species
are also interesting themes to investigate. Mangrove plants
offer both theoretical and biotechnological potential for
improving plant growth in high salt environments, how-
ever, information on their physiology, biochemistry, and
molecular biology remains limited. The development of
in vitro culture systems is important for complementation
of whole plant studies. In 2001, the successful establish-
ment of a suspension culture from leaf explants of Bru-
guiera sexangula (Rhisophoraceae) was reported [7,8].
Protoplast fusion studies [9] and molecular biological
work on salt-tolerance related genes has since been per-
formed [10]. In order to expand our knowledge of man-
groves, we recently established a suspension culture from
cotyledon explants of the mangrove, Sonneratia alba
(Sonneratiaceae), with subsequent study of its organo-
genic potential [11]. The effects of four main salt ingre-
dients of sea water on S. alba were investigated and its
halophilic nature and tolerance to NaCl were determined.
They were compared with suspension cultures of B. sex-
angula, and tobacco BY-2 cells [12]. S. alba grows at the

Effects of Sea Salts on Induction of Cell Proliferation in Liquid Cultures of Mangrove plants,
Sonneratia caseolaris and S. alba
36
most seaside coast of the Iriomote Island, Okinawa, Ja-
pan [13], while B. sexangula grows in more in-land areas
of Thailand [14].
To investigate the mechanisms of salt tolerance, a
comparison of salt-tolerant and salt-intolerant plants, or
cells of similar genetic background but different salt tol-
erance, is required. The latter investigation is problematic,
because obtaining a specific tree mutant is very difficult.
In the last few years, we have studied another mangrove
plant, S. caseolaris, which is in the same genera as S.
alba and shares its habitat in the in-land area of man-
grove forests of Myanmar and Thailand [14]. Some cell
proliferation was obtained from cotyledons and hypocot-
yls of S. caseolaris using the same hormonal conditions
as for S. alba [11].
The objective of this study was to investigate the cell
proliferation of different types of explants from S. case-
olaris in response to KCl, NaCl, CaCl2, MgCl2, MgSO4,
which are components of sea water. The results were
compared with cell proliferation from S. alba cotyledon
explants. One potential effect of adding salts to a culture
system is the osmotic effect on cells. In order to assess
the importance of osmotic potential on the proliferation
of mangrove cells, several osmotically active compounds,
sorbitol, mannitol and glycinebetaine, which are com-
monly used in tissue systems, were also tested. A small-
scale liquid culture method [11] was used which mini-
mized the volume of plant material required.
2. Materials and Methods
2.1. Materials
Fruits of S. caseolaris were collected at the Khu Khut,
Songkla or at Khanom river Basin, Si Thammarat Prov-
ince, Thailand, and those of S. alba were collected from
Iriomote island, Okinawa, Japan. Aseptic procedures for
S. alba were replicated from Kawana et al. [11]. Seeds of
S. caseolaris were disinfected by treatment with deter-
gent, soaked in 70% ethanol solution for 1 min, washed
with water, soaked in 3% sodium hypochlorite solution
(NaClO) for 1 hr, and finally washed with sterile water
three times.
Disinfected seeds were germinated on 5 ml of 0.8%
w/v agar medium (tissue culture grade, Wako Chemicals
Co. Ltd) in 15-ml test tubes under a 16 hr photoperiod
(80 μmol m-2s-1), at 24˚C. After two to five months of
culture, sterile hypocotyls and cotyledons were cut into
two to three, 1 mm-wide sections for culture.
As leaves did not develop in sterile culture, additional
seeds were planted in a mixture of sand, vermiculite and
soil in 1/10000a Wagner’s pots (ICW-2, ICM Co. Ltd,
Tsukuba, Japan) in a container and watered weekly. The
plants of S. caseolaris were maintained in a green house
in natural light conditions at 15-45˚C. After six months
to three years of growth, the top four leaves of each plant
were harvested. Leaves were washed with water and de-
tergent, sterilized with 1% NaClO for 30 min to 1 hr and
washed with sterile water three times. They were then cut
into 1 mm-wide sections in a manner similar to that for
the cotyledons.
2.2. Liquid Culture
Disinfected cotyledons of S. caseolaris and S. alba, as
well as hypocotyls and leaves of the former were cut in a
Petri dish under axenic conditions. Two or three sections
were placed in a 10 mL flat-bottomed culture tube (Ma-
ruemu No.3) with 1 mL liquid Murashige & Skoog’s
(MS) [15] basal medium containing 0.1 μM 2,4-di-chlo-
rophenoxyacetic acid (2,4-D) and 3% w/v of sucrose or
glucose (when cited in text), and covered with autoclaved
translucent film (BioHazard Bag 86.1199, Assist Co.
Ltd.). The pH was adjusted to 5.8 before sterilization at
121˚C, 20 min. All cultures were incubated in the dark at
30˚C on a rotary shaker at 100 rpm. When cited in the
text, 10, 12.5, 25, 50, 100, or 200 mM of KCl, NaCl,
CaCl2, MgCl2, or MgSO4 was added to the above media.
Sorbitol, mannitol or glycinebetaine at 25, 50, 100, 200
or 400 mM were also added. The cultures were observed
under an inverted microscope (Olympus CK40).
2.3. Data Description
In S. caseolaris, two methods of quantifying the degree
of cell proliferation were used. First, the total area of
proliferated mass per explant was calculated using the
image analysis program, Image J [16] to analyze digital
photographs taken through the inverted microscope (Fig.
1). As this method is limited to a single two-dimensional
representation of each cell mass, we also described cell
proliferation at the cut surface of the explants in terms of
a system of grades based on direct observation under an
inverted microscope. In S. caseolaris, Grado 0, flat sur-
face with no reaction; Grade 1, one cell layer appeared
from the cut surface; Grade 2, two or three cells layers
were present; Grade 3, 4-5 layers of proliferated cells
could be found on the cut surface but the area of prolif-
eration was more localized; Grade 4, 4-5 cells layer of
proliferated cells covering a larger area of the cut surface;
and Grade 5, more proliferation than 4. In S. alba, prolif-
eration from both cut surfaces was assessed in assigning
the numbers of grade (0, 1-4). All data were averaged per
explant from data of 4 to 8 explants in 2 to 4 tubes. A
student t-test was performed on the difference of mean
values between the control (i.e. without added salts and
osmotic compounds) and each treatment at P < 0.05.

Effects of Sea Salts on Induction of Cell Proliferation in Liquid Cultures of Mangrove plants,
Sonneratia caseolaris and S. alba
37
3. Results
In the liquid culture of S. caseolaris, cell proliferation
was observed at the cut surface of sections under an in-
verted microscope (Figures 1(a)-(c)). A freehand-traced
area of cell proliferation (Figure 1(b), (c)) was measured
from the photographs taken of each explant using the
Image J program.
The effects of KCl, NaCl, CaCl2, and MgCl2 on cell
proliferation from leaves (Figure 2) and cotyledons (Fig-
ure 3) of S. caseolaris were investigated. The total area
of proliferation in controls was 0.63 mm2/leaf explant
(Figure 2) and 0.53 mm2/ cotyledon explant (Figure 3).
Low concentrations of MgCl2 (12.5 mM and 25 mM)
applied to leaves stimulated cell proliferation approxi-
mately 1.5-2.5-fold. No increase in cell proliferation was
observed in leaves treated with KCl, NaCl and CaCl2. In
cotyledons, concentrations of MgCl2, from 12.5 to 100
mM, increased cell proliferation approximately 2-3-fold
over the control, while no clear stimulation was observed
by KCl, NaCl and CaCl2. All four salts tested were in-
hibitory at 200 mM (Figure 3).
Asterisk on the top of bars in Figures 3 to 7 showed
that differences were found at p < 0.05 by t-test. From
the mean values of grade of reaction, percentages of
stimulation or inhibition compared to zero control were
calculated.
Effects of KCl, NaCl, CaCl2 and MgCl2 on cell prolif-
eration from cotyledons of S. alba in a liquid culture
were investigated (Figure 4). At zero concentration (the
control of no additional salts), the grade of reaction was
2.6 ± 0.28 (S.E.)/explant. Low concentration (10 mM) of
KCl strongly (80%) stimulated cell proliferation. No in-
hibition was observed at a wide range of NaCl concen-
trations (10-50 mM). Stimulation by MgCl2 was not ob-
served and inhibition by CaCl2 was prominent. All salts
at 200 mM totally inhibited growth.
Effects of KCl, NaCl, CaCl2, MgCl2 on cell prolifera-
tion from hypocotyls of S. caseolaris were investigated
(Figure 5). The grade of reaction of the zero control was
2.3 ± 0.25 (S.E.)/hypocotyl explant. Low concentrations
of MgCl2 (12.5-25 mM) stimulated cell proliferation
(44-111%), in a manner similar to that of cotyledons of S.
caseolaris (Figure 3) but different from cotyledons of S.
alba, in which MgCl2 did not stimulate cell proliferation
(Figure 4). Intermediate concentrations (25-50 mM) of
NaCl (44-67%) and CaCl2 (44-56%) stimulated cell pro-
liferation. Tolerance at 200 mM NaCl was also observed.
Since magnesium ions were effective in causing cell
proliferation in S. caseolaris leaf explants (Figure 2),
two different magnesium salts, i.e. MgCl2 and MgSO4,
were tested to assess the effects of anions (Figure 6).
(a)
(b)
(c)
Figure 1. Measurement of the area of cell proliferation in
liquid cultures of S. caseolaris leaf explant. (a) Cut surface
before culture. (b) Proliferation of cells from the cut surface.
(c) Area of proliferation from (b) was freehand-traced and
its area was measured using the image analysis program,
Image J. bar = 250 μm.

Effects of Sea Salts on Induction of Cell Proliferation in Liquid Cultures of Mangrove plants,
Sonneratia caseolaris and S. alba
38
Figure 2. Effects of KCl, NaCl, CaCl2 and MgCl2 on induc-
tion of cell proliferation from leaves of S. caseolaris. Data
were area of proliferation. Days of culture were 32.
Figure 3. Effects of KCl, NaCl, CaCl2 and MgCl2 on cell
proliferation from cotyledons of S. caseolaris. Data were
area of proliferation. Days of culture were 34.
Both formats of data description (area of proliferation in
Figure 6(a) and the grade of proliferation in Figure 6(b))
showed similar results, except for the reaction to MgSO4
at 200 mM. At zero control, the area of proliferation was
1.03 ± 0.29 (S.E.) mm2/explant (Figure 6(a)) and num-
ber of grade was 1.9 ± 0.31 (S.E.)/explant (Figure 6(b)).
Low concentrations (12.5-25 mM) of MgCl2 stimulated
cell proliferation from leaf explants, though the percent-
age of stimulation (70%) was lower than that of Figure 2,
while less stimulation (23-50%) was obtained with MgSO4.
High concentrations (100-200 mM) of MgCl2 were in-
hibitory, however, no inhibitory effect was observed at
up to 100 mM of MgSO4. When 3% glucose was used
Figure 4. Effects of KCl, NaCl, CaCl2 and MgCl2 on cell
proliferation from cotyledons of S. alba. Data were the
grade of proliferation. Days of culture were 34.
Figure 5. Effects of KCl, NaCl, CaCl2 and MgCl2 on cell
enlargement and proliferation from hypocotyls of S. caseo-
laris. Data were the grade of proliferation. Days of culture
were 48.
instead of sucrose in the medium, a clear stimulation
(58-163%) by low to intermediate concentrations (12.5-
50 mM) of MgCl2 and tolerance at high concentration of
MgSO4 were also observed (Figure 6(c)). The number of
grade of proliferation was 1.6 ± 0.19 (S.E.)/explant with-
out additional salts.
The effects of different osmotic compounds in liquid
cultures on leaves of S. caseolaris were investigated (Fig-
ure 7). The grade of reaction at zero control was 2.3 ±
0.4 (S.E.)/leaf explant. The cultures could tolerate con-
centrations up to 100 mM. However, no clear stimulation
of growth was observed. Both sorbitol and mannitol be-

Effects of Sea Salts on Induction of Cell Proliferation in Liquid Cultures of Mangrove plants,
Sonneratia caseolaris and S. alba
39
Figure 6. Comparison of the effects of MgCl2 and MgSO4 on
cell proliferation from leaves of S. caseolaris. The control
medium is MS containing 0.1 μM 2,4-D and 3% sucrose (a,
b) and 3% glucose (c). Days of culture were 68. Data were
the area of proliferation (a) and grade of proliferation (b,
c).
gan to produce significant inhibitory effects at 200 mM
and they became totally inhibitory to growth at 400 mM.
When the effect of sorbitol in combination with 3% su-
crose was investigated in liquid culture of cotyledons
(Figure 8), the grade of reaction at zero control was 2.4 ±
0.26 (S.E.)/cotyledon explant. Tolerance up to 200 mM
of sorbitol was observed and 400 mM sorbitol was in-
hibitory. The pattern is very different from those of the
four salts, i.e. KCl, NaCl, MgCl2 and CaCl2. Glycinebe-
taine totally inhibited growth in cultures of cotyledons
and leaves of S. caseolaris at any concentrations (data
not shown).
Figure 7. Effects of sorbitol and mannitol in combination
with 3% sucrose on cell proliferation from leaves of S. case-
olaris. Days of culture were 35. Data were the grade of pro-
liferation.
Figure 8. Effect of sorbitol in combination with 3% sucrose
on cell proliferation from cotyledons of S. caseolaris. Data
were the grade of proliferation. Days of culture were 37.
4. Discussion
4.1. Differences in Response to Added Salts
between S. caseolaris and S. alba
Tolerance to a wide range (10-50 mM) of concentrations
of NaCl was observed in cotyledon cultures of S. alba
but not in cotyledon cultures of S. caseolaris. The fact
that S. alba grows along the most seaside coast while S.
caseolaris grows more in-land, may have contributed to
the different responses observed. The salinity of the latter
growing area was lower than that of the former [17]. In
the suspension culture derived from cotyledons of S. alba,
stimulation of growth was observed by NaCl at the same
concentrations [12]. S. alba would have developed
mechanisms to deal with the key component of sea water,
i.e. sodium ions in order to achieve proper growth and
development. Sea water and the mangrove growing soil
contain substantial amount of other salts [18]. Induction

Effects of Sea Salts on Induction of Cell Proliferation in Liquid Cultures of Mangrove plants,
Sonneratia caseolaris and S. alba
40
of cell proliferation was greatly stimulated by KCl in
cotyledon culture of S. alba but not in any of the S. case-
olaris tissues. In the established suspension culture of S.
alba, increase of growth was obtained at low concentra-
tion (10 mM) of KCl [12]. On the contrary, low concen-
trations (12.5-25 mM) of MgCl2 greatly stimulated cell
induction from all three tissues in S. caseolaris, but not
in the cotyledons of S. alba. No data of hypocotyls or
leaves of S. alba were available in this report, because
the growth of big seedlings was difficult in S. alba. To
explore the mechanisms of salts tolerance, the develop-
ment of in vitro culture systems is important for com-
plementation of whole plant studies. S. alba and S. case-
olaris are two excellent mangrove species of the same
genera showing different responses to different salts.
These can be cultured in the same MS basal medium
containing 3% of sucrose and 0.1 μM of 2,4-D, while
modified amino acid basal medium was employed for the
culture of B. sexangula of different family [8].
4.2. Differences in Tissue Responses to Salts in
S. caseolaris
Differences were found in the reaction to different salts
depending on the source of tissue tested, i.e. leaves,
cotyledons, or hypocotyls. Stimulation by NaCl was ob-
served in cultures of hypocotyls, but not in cotyledons or
leaves. Similarly, CaCl2 was stimulatory to hypocotyl
culture but had little effect on other tissues of S. caseo-
laris. Cotyledons and hypocotyls were more tolerant to
MgCl2 than leaves. In nature, young seedlings of Son-
neratia species sometimes grow under water but leaves
are rarely submerged. Thus, we would expect cotyledons
and hypocotyls to be more tolerant tissues to salty water
than leaves. The observed difference in tolerance to NaCl
depending on the origin of tissues, i.e. viviparous seed-
lings and leaves, was also reported for callus induction of
B. sexangula [7].
Effects of salts can be explained partly as the osmotic
effects of component ions. However, MgCl2 and CaCl2
were not always inhibitory compared to NaCl and KCl
[12], implying that cations themselves are important in
mangrove cells.
This is the first report where effects of high concentra-
tions of MgSO4 were investigated, and tolerance of
leaves of S. caseolaris was found in liquid culture, while
low concentrations of MgSO4 were not stimulatory com-
pared with MgCl2. Tolerance to MgSO4 was also found
in media containing glucose (Figure 6(c)), which sugar
stimulated cell proliferation from leaves of S. caseolaris
in solid culture [19]. Although differences in ion disso-
ciation can result in different osmotic potentials, the re-
sults in Figure 6 suggest anions may have specific roles
to play. Both anions are ingredients of sea water and fur-
ther studies are needed to ascertain their function.
4.3. Effects of Osmotic Compounds in
S. caseolaris
The presence of salts naturally found in sea water can
change the osmotic environment surrounding plants.
Changes in the osmotic environment have a substantial
effect on plant growth and development. It has been
shown that osmotic stress enhances somatic embryo for-
mation in carrot [20] and mannitol can induce somatic
embryogenesis in vegetative tissues of Arabidopsis [21].
In this study, several common osmotica were used to
assess the importance of the osmotic environment on cell
proliferation of mangrove species. As shown in Figure 7,
neither mannitol nor sorbitol had a significant stimula-
tory effect on cell proliferation of S. caseolaris, and be-
came inhibitory to growth at 200 mM. In leaf culture of a
Rhizophoraceae mangrove, Rhizophora stylosa, sorbitol
at 0.2-0.4 M was stimulatory for callus induction, while
NaCl was inhibitory at the same osmotic potential [22].
Mannitol and glycinebetaine are known as naturally oc-
curring osmotica in S. alba [23] and an Avicenniaceae
mangrove, Avicennia marina [24], respectively. The gly-
cinebtaine had a negative effect on growth even at low
concentrations (data not shown). These suggest that
changes in osmotic conditions alone are not sufficient to
promote growth. The effects of salts cannot be described
only by their effects as osmoticum. Both cations and
anions of various salts may have specific functions to
play in promoting growth and development of mangrove
plants.
4.4. Data Description Method
In this report, in addition to recording the response as the
numbers of grade of proliferation, which was used in the
previous report in culture of S. alba [11], we introduced
another data description method, the area of cell prolif-
eration measured using an image analysis program. No
critical difference in the effects of salts was found be-
tween two methods of data description. Although both
methods yielded similar results, the grade method is pre-
ferred as one can identify small changes in the explant,
especially at high salt concentrations, and the standard
errors were smaller than the area method. The area
method can only account for two dimensional changes in
an explant and the process is time consuming. The use of
small volume culture method as detailed in this study
enables the assessment of changes of explants to differ-
ent tested variables. Since the sources of seed materials
are limited, this greatly increases the efficiency of the
study.

Effects of Sea Salts on Induction of Cell Proliferation in Liquid Cultures of Mangrove plants,
Sonneratia caseolaris and S. alba
41
4.5. Conclusions
Mangrove plants have adapted to grow in an environ-
ment with high salts. This unique property is also re-
flected in the cell cultures generated from the mangrove
species, Sonneratia caseolaris and S. alba. The ability of
mangrove cell cultures to grow in the presence of high
salts indicates the unique adaptation and metabolism of
mangrove cells. It is important to note that different sea
water components, KCl, NaCl, CaCl2, MgCl2 and MgSO4,
elicit different responses from different mangrove spe-
cies. This result indicates that different mangrove species
have different metabolic adaptations to various salts in
the environment. The ability of both mangrove species to
tolerate high levels of magnesium ions indicates that
magnesium ions may have a vital metabolic role to play
within mangrove cells. Cell cultures generated from dif-
ferent tissues of the same plant react differently to vari-
ous salts and salt concentrations. This observation sug-
gests that different tissue types within the plant body
responds to salts differently. Future biochemical and cell
biological studies will provide further insight into the
role of various ions on the growth of mangrove cultures.
5. Acknowlegements
Authors thank Prof. K. Suzuki of the Yokohama National
University for his kind supply of the fruits of S. caseo-
laris. Authors also thank Prof. E. C. Yeung of the Uni-
versity of Calgary for his critical reading of this manu-
script.
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